Hybrid breeding methods for crop plants in the family brassicaceae

ABSTRACT

This invention relates to a hybrid breeding technology for crop plants in the family Brassicaceae characterized in that F 1  seed is produced by crossing the female parent of a male sterile line introduced self-incompatibility with the male parent of a self-incompatible line. By the breeding technology of this invention, selfed seeds contamination can be prevented in F 1  breeding and F 1  seed production of crop plants in the family Brassicaceae and, moreover, the cost of seed production can be reduced through an improved seed production efficiency.

BACKGROUND OF THE INVENTION

This invention relates to a hybrid breeding method for crop plants in the family Brassicaceae.

The invention relates to a breeding method for an F₁ variety which, particularly in rape, is double-low (low erucic acid-low glucosinolate content) and improved in yield, oil content and quality, and disease and pest resistance.

Referring to rapeseed (Brassica napus, n=19), which is self-compatible, the utilization of F₁ has not been made to this day partly because the production of F₁ seed through utilization of self-incompatibility is not feasible and partly because a stable male sterile line which is not affected by temperature or day length and the fertility restoring gene for the male sterility have not been discovered as yet. As to other crop plants in the family Brassicaceae, too, several plants and varieties are unstable in the expression of self-incompatibility in the production of F₁ by the utilization of self-incompatibility and there is the problem that F₁ is occasionally contaminated with selfed seeds (hereinafter referred to as "intra") as well as the problem that the cost of F₁ seed production inclusive of the cost of bud pollination for parent seed production is high.

Meanwhile, labor conservation is a major objective in the fields of stock seed production, F₁ seed production, seed cleaning and cultivation and, as one aspect of this recent trend, the requirement in regard to the purity of seed is getting more and more stringent.

SUMMARY OF THE INVENTION

The object of this invention is to provide a hybrid breeding method of improved efficiency which helps to prevent intra contamination and contributes to cost reduction in the production of F₁ seed from crop plants in the family Brassicaceae.

For the F₁ breeding of rape, the inventors of this invention envisaged the development of lines possessing stable male sterility and fertility restoring genes for male sterility and the introduction of self-incompatible genes from related species and conducted a large amount of research. As a result, the inventors discovered a combination of cytoplasm with male sterility showing a very stable expression of male sterility and fertility restoration with a fertility restoring gene and utilizing the combination developed an F₁ rape variety No. 9122! of spring type which is double-low and promises an increased yield. Furthermore, for cost reduction through increased seed yield, the inventors developed an F₂ variety of said rape, namely T-410!. The inventors further developed an F₁ variety No. 9123!, using a new female parent derived from a B line which was excellent in disease resistance and seed production efficiency. In addition, by introducing several kinds of self-incompatible genes, the inventors succeeded in the development of rape lines possessing various excellent characters. Then, the inventors did further research for the prevention of intra contamination in the production of F₁ seed and the reduction of seed production cost and have developed a highly efficient hybrid breeding method for crop plants in the family Brassicaceae, which is based on a combination of male sterility and self-incompatibility (the selection and development of lines compatible with carbon dioxide treatment).

A first hybrid breeding process for crop plants in the family Brassicaceae in accordance with this invention is characterized in that F₁ seed is produced by crossing the female parent of a male sterile line introduced self-incompatibility with the male parent of a self-incompatible line (FIG. 13).

This process is most effective for the prevention of intra contamination associated with unstable self-incompatibility and a large difference in flowering time between the parents, among other causes. When the self-incompatibility of the male parent is unstable, the male parent is cut off and the seed is not harvested from the male parent for the prevention of intra contamination. When the self-incompatibility of the male parent is stable, there is no seed formation on the male parent so that the male parent need not be cut off but both the male and female parents can be reaped indiscriminately, with the result that a remarkable cost reduction is realized. Thus, mix-sowing of male and female parents, mechanical sowing and mechanical harvesting are made possible. This process is useful for radish, cabbages and Chinese cabbages.

A second process according to this invention is characterized in that F₁ seed is obtained by crossing the female parent of a male sterile line introduced self-incompatibility with the male parent of a self-incompatible or self-compatible line possessing fertility restoring gene (FIG. 17).

The first process is not suitable for the breeding of crop plants which their seeds were utilized such as rape, for F₁ shows sterility and self-incompatibility. The second process, which overcomes this drawback, is characterized in that a fertility restoring gene for restoration of pollen fertility in F₁ and a self-incompatible or self-compatible gene are introduced into the male parent. While the advantages of this second process are similar to those of the first process, it has the additional advantage that because of the consequent restoration of pollen fertility, the utilization of F₂ becomes feasible, in particular, and a still greater seed yield and a more remarkable cost reduction can be realized. Particularly the reduction of seed production cost is a matter of top priority in rape F₁ breeding and this process as well as a fourth process to be described below is a very effective technique and these processes can be selectively used according to the characteristics and F₁ combining abilities of the lines. This process is particularly effective for the utilization of F₂ of crop plants in the family Brassicaceae.

A third process of this invention is characterized in that F₁ seed is obtained by crossing the female parent of a male sterile line introduced self-compatibility with the male parent of a self-compatible line or the male parent of a self-incompatible line (FIG. 32).

This process is effective for crop plants having no self-incompatibility or crop plants which are dominantly self-incompatible but have strong self-compatibility or for the development of F₁ varieties of these lines. For example, this process is effective for karashina (mustard plant), takana (leaf mustard), radish, cabbages, Chinese cabbages and so on.

A fourth process according to this invention is characterized in that F₁ seed is obtained by crossing the female parent of a male sterile line introduced self-compatibility with the male parent of a self-incompatible line possessing fertility restoring gene (FIG. 36).

In the second process mentioned above, the female parent is self-incompatible and male sterile but when a self-compatible and male sterile line is used as the female parent as in this process, the F₁ breeding of highly self-compatible crops and varieties, reduction of F₁ seed production cost, and improved F₂ seed production efficiency can be realized. Particularly, when the number of self-incompatible genes is increased for four-way crossing, the effect of open flower crossing in the utilization of F₂ is remarkable and the seed production capacity is increased. This process is effective for rape in the main. Moreover, even in single crossing or three-way crossing, too, the incorporation of a self-incompatibility in a male parent possessing fertility restoring gene dispenses with the need for cutting off the male parent, thus enabling omnibus reaping. The method of crossing can be selected according to the characteristics and F₁ combining ability of the lines.

In accordance with the breeding technology of this invention, intra contamination in the F₁ breeding and F₁ seed production of crop plants in the family Brassicaceae can be prevented and, moreover, the cost of seed production can be reduced through an improved seed production efficiency.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation of the breeding process according to an embodiment of this invention;

FIG. 2 is a diagrammatic representation of the breeding process according to another embodiment of this invention;

FIG. 3 is a diagrammatic representation of the breeding process according to still another embodiment of this invention;

FIG. 4 is a diagrammatic representation of the breeding process according to still another embodiment of this invention;

FIG. 5 is a diagrammatic representation of the breeding process according to still another embodiment of this invention;

FIG. 6 is a diagrammatic representation of the breeding process according to still another embodiment of this invention;

FIG. 7 is a diagrammatic representation of the breeding process according to still another embodiment of this invention;

FIG. 8 is a diagrammatic representation of the breeding process according to still another embodiment of this invention;

FIG. 9 is a diagrammatic representation of the breeding process according to still another embodiment of this invention;

FIG. 10 is a diagrammatic representation of the breeding process according to still another embodiment of this invention;

FIG. 11 is a diagrammatic representation of the breeding process according to still another embodiment of this invention;

FIG. 12 is a diagrammatic representation of the breeding process according to still another embodiment of this invention;

FIG. 13 is a diagrammatic representation of the breeding method according to an embodiment of this invention;

FIG. 14 is a diagrammatic representation of the breeding process according to still another embodiment of this invention;

FIG. 15 is a diagrammatic representation of the breeding process according to still another embodiment of this invention;

FIG. 16 is a diagrammatic representation of the breeding process according to still another embodiment of this invention;

FIG. 17 is a diagrammatic representation of the breeding method according to another embodiment of this invention;

FIG. 18 is a diagrammatic representation of a part of the breeding process according to still another embodiment of this invention;

FIG. 19 is a diagrammatic representation of a part of the breeding process according to the same embodiment corresponding to FIG. 18, which is sequential to the bottom of FIG. 18;

FIG. 20 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 18 and 19, which is sequential to the right of FIG. 18;

FIG. 21 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 18-20, which is sequential to the bottom of FIG. 20 and the right of FIG. 19;

FIG. 22 is a diagrammatic representation of a part of the breeding process according to still another embodiment of this invention;

FIG. 23 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIG. 22, which is sequential to the bottom of FIG. 22;

FIG. 24 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 22 and 23, which is sequential to the right of FIG. 22;

FIG. 25 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 22-24, which is sequential to the bottom of FIG. 24 and the right of FIG. 23;

FIG. 26 is a diagrammatic representation of a part of the breeding process according to still another embodiment of this invention;

FIG. 27 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIG. 26, which is sequential to the bottom of FIG. 26;

FIG. 28 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 26 and 27, which is sequential to the right of FIG. 26;

FIG. 29 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 26-28, which is sequential to the bottom of FIG. 28 and the right of FIG. 27;

FIG. 30 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 26-29, which is sequential to the right of FIG. 28;

FIG. 31 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 26-30, which is sequential to the bottom of FIG. 30 and the right of FIG. 29;

FIG. 32 is a diagrammatic representation of the breeding method according to still another embodiment of this invention;

FIG. 33 is a diagrammatic representation of the breeding process according to still another embodiment of this invention;

FIG. 34 is a diagrammatic representation of the breeding process according to still another embodiment of this invention;

FIG. 35 is a diagrammatic representation of the breeding process according to still another embodiment of this invention;

FIG. 36 is a diagrammatic representation of the breeding method according to still another embodiment of this invention;

FIG. 37 is a diagrammatic representation of a part of the breeding process according to still another embodiment of this invention;

FIG. 38 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIG. 37, which is sequential to the bottom of FIG. 37;

FIG. 39 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 37 and 38, which is sequential to the right of FIG. 37;

FIG. 40 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 37-39, which is sequential to the bottom of FIG. 39 and the right of FIG. 38;

FIG. 41 is a diagrammatic representation of a part of the breeding process according to still another embodiment of this invention;

FIG. 42 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIG. 41, which is sequential to the bottom of FIG. 41;

FIG. 43 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 41 and 42, which is sequential to the right of FIG. 41;

FIG. 44 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 41-43, which is sequential to the bottom of FIG. 43 and the right of FIG. 42;

FIG. 45 is a diagrammatic representation of a part of the breeding process according to still another embodiment of this invention;

FIG. 46 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIG. 45, which is sequential to the bottom of FIG. 45;

FIG. 47 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 45 and 46, which is sequential to the right of FIG. 45;

FIG. 48 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 45-47, which is sequential to the bottom of FIG. 47 and the right of FIG. 46;

FIG. 49 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 45-48, which is sequential to the right of FIG. 47;

FIG. 50 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 45-49, which is sequential to the bottom of FIG. 49 and the right of FIG. 48.

In FIGS. 1-4, FIGS. 10-12, FIGS. 14-16, FIGS. 18-31, FIGS. 33-35 and FIGS. 37-50, ∘ stands for F (male fertility). In FIGS. 5-9, ∘ stands for an individual. In FIGS. 1-12, FIGS. 14-16, FIGS. 18-31, FIGS. 33-35 and FIGS. 37-50, ∘ stands for MS (male sterility), x for Cross (crossing), Δ for r (fertility restoring gene), ,  and ▴ for test individuals, □ for individual seed production, ▪ for mass seed production, S^(a), S^(b), S^(d) and S^(e) for self-incompatible genes. In FIGS. 13, 17, 32 and 36, S₁, S₂, S₃ and S₄ stand for self-incompatible genes, S_(f1) and S_(f2) for self-compatible genes, ms for cytoplasm with male sterility, r for fertility restoring gene, and * for seed production by CO₂ treatment or bud pollination.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The breeding processes according to this invention and the advantages of the breeding technology of the invention are now described in detail with reference to examples.

1. Breeding of rape F₁ variety No. 9122! by the utilization of male sterility (FIGS. 1-3)

Breeding process: The F₁ obtained by the utilization of male sterility was slightly unstable in the expression of male sterility in the winter variety and was difficult to breed. Therefore, the breeding of a spring variety with stable male sterility was attempted.

1) Development of AB line 60To-AB! (FIG. 1)

A selected line 60To! could be developed as a maintainer for the male sterile line MS-N1! discovered from among N-1!s which were spring varieties in 1987. This was later made 60To-B!, subjected to continuous backcrossing, individual selection during 5 generations and, then, mass selection. Selections were carried out, with emphasis on spring growing habit and double-low feature, in regard to the size and shape of the pod, plant posture and disease resistance, among others.

2) Development of C line 62WeB-C! (FIG. 2)

The fertility restoring gene for MS-N1!-derived cytoplasm with male sterility was discovered in the winter variety IM line! and named IM-B!. In 1988, this was crossed with a spring double-low line 62We!. Then, with the homozygotic presence of a fertility restoring gene being confirmed, selection breeding was carried out, with emphasis on spring growing habit and double-low feature, in regard to the size and shape of the pod, plant posture, and alignment in flowering time with the male sterile AB line on the female parent side.

3) Development of F₁ No. 9122! (FIG. 3)

By testing a number of F₁ combinations, the parent lines with the highest combining ability were selected from said AB and C lines and F₁ No. 9122! was developed.

The result of investigation of the seed yield of this F₁ No. 9122! is shown in Table 1. It is apparent from the table that the seed yield of F₁ No. 9122! in 1992 was fairly high as compared with the control variety OAC Triton!. The increased seed yield of F₁ No. 9122! contributes to a reduced cost of seed production of F₂ T-410!.

                                      TABLE 1                                      __________________________________________________________________________     Comparison of seed yields of F.sub.1  No. 9122! and control rape               Takii Plant Breeding and Experiment Station, Kosei-cho, Koka-gun, Shiga        Prefecture                                                                     (Sowing: November 25, 1991; investigation: July 3, 1992)                                                           Yield,                                                                               Seed                                      Plant-      Amount of                                                                           Flowering                                                                           Flowering                                                                               l/10 a                                                                               produc-                              Spring,                                                                             ing                                                                               Number                                                                             Seed seed per                                                                            began                                                                               ended                                                                               Degree                                                                             (on a 40                                                                             tion                                 1992 area                                                                              of  produced                                                                            plant                                                                               (month/                                                                             (month/                                                                             of bee                                                                             thousand                                                                             index                                Variety                                                                             (m.sup.2)                                                                         plants                                                                             (l)  (ml) date)                                                                               date)                                                                               visit                                                                              plant basis)                                                                         (%)                                  __________________________________________________________________________     No. 9122                                                                            3.75                                                                              143 1.8  12.6 4/16 5/20 Excel-                                                                             504   171                                  (F.sub.1)                       lent                                           OAC  7.05                                                                              272 2.0  7.4  4/17 5/20 Excel-                                                                             294   100                                  Triton                          lent                                           (common                                                                        variety)                                                                       __________________________________________________________________________

Table 2 shows the glucosinolate contents and fatty acid compositions of F₁ No. 9122!, main Canadian varieties (3 varieties), registered varieties Asaka-no-natane (registration no. Natane Norin 46) and Kizaki-no-natane (registration no. Natane Norin 47) developed at Tohoku Agricultural Experiment Station. Asaka-no-natane and Kizaki-no-natane, both of which are domestic varieties, are close to the international level in erucic acid content but are by far higher in glucosinolate content, namely, single-low. In contrast, F₁ No. 9122! can be regarded as a double-low line with its glucosinolate and erucic acid contents being both comparable to the international levels.

                                      TABLE 2                                      __________________________________________________________________________     The glucosinolate contents and fatty acid compositions of F.sub.1  No.         9122! and control cultivars 1992                                                      Gluco-                                                                         sino-                                                                              Fatty acid (%)                                                             late                                                                               Myrist-                                                                            Palmit-                                                                            Stear-                                                                             Ole-                                                                               Linol-                                                                             Linolen-                                                                            Arachidon-                                                                           Eicosan-                                                                            Behen-                                                                             Eruc-                       Cultivar                                                                              (μM/g)                                                                          ic acid                                                                            ic acid                                                                            ic acid                                                                            ic acid                                                                            ic acid                                                                            ic acid                                                                             ic acid                                                                              ic acid                                                                             ic acid                                                                            ic acid                     __________________________________________________________________________     No. 9122.sup.1)                                                                       16.8                                                                               0.0 5.0 1.5 63.7                                                                               20.4                                                                               7.7  0.5   1.2  0.0 0.0                         Tobin.sup.2)                                                                          20.1                                                                               0.0 3.4 2.4 56.1                                                                               24.4                                                                               11.6 0.5   1.5  0.0 0.0                         OAC Triton.sup.2)                                                                     15.8                                                                               0.0 3.9 2.6 54.4                                                                               22.0                                                                               9.6  0.6   2.9  0.3 3.6                         Westar.sup.2)                                                                         17.0                                                                               0.0 3.9 2.8 61.6                                                                               20.5                                                                               7.8  0.6   2.0  0.3 0.5                         Asaka-no                                                                              >50     4.6     61.2                                                                               21.9                                                                               8.7        1.3      0.2                         natane.sup.3)                                                                  Kizaki-no                                                                             >50     4.7     63.7                                                                               18.8                                                                               8.9        1.3      0.1                         natane.sup.3)                                                                  __________________________________________________________________________      .sup.1) Assayed by Nippon Oil and Fat Testing Association (glucosinolate       contents were determined at Takii Plant Breeding and Experiment Station)       .sup.2) Determined at Takii Plant Breeding and Experiment Station              .sup.3) Assayed by Tohoku Agricultural Experimental Station              

2. Breeding of rape F₁ No. 9123! by the utilization of male sterility (FIG. 4)

For further enhancement of disease resistance, lodging resistance and seed yield of the F₁ variety No. 9122!, a new maintainer (B line) was developed for the breeding of F₁ variety No. 9123!. The maintainer was 2DR-B! obtained by the serial selection and breeding carried out since 1990.

Table 3 shows the seed yield data for F₁ No. 9122!. Table 4 shows the seed yield data for F₁ No. 9123!. In the spring of 1992, a field trial of F₁ No. 9122! was carried out in Canada. Then, in the spring of 1993, field trials of F₁ No. 9122! and No. 9123! were carried out in the Netherlands.

The comparison of seed yields of F₁ No. 9122! and F₁ No. 9123! is presented in Table 5.

                                      TABLE 3                                      __________________________________________________________________________     Seeds yields of rape F.sub.1  No. 9122!                                        Takii Naganuma Breeding Station, Naganuma-cho, Yubari-gun, Hokkaido,           1991-1992                                                                                   1991           1992                                                               Seed           Seed                                               Line      Area                                                                              produced    Area                                                                              produced                                        Line                                                                              combination                                                                              (m.sup.2)                                                                         (l)  Remarks                                                                               (m.sup.2)                                                                         (l)  Remarks                                    __________________________________________________________________________     AB 60To-AB × 60To-B                                                                   20 10.0        230                                                                               30.0                                            B  60To-B    10 5.7         87 15.0                                            C  62WeB-C   18 6.5         60 20.0                                            F.sub.1                                                                           60To-AB × 62WeB-C                                                                  50 17.0 Field trial                                                                           460                                                                               50.0 Field trial in                                                  in Canada      the Netherlands                                                 (spring, 1992) (spring, 1993)                             __________________________________________________________________________

                                      TABLE 4                                      __________________________________________________________________________     Seeds yields of rape F.sub.1  No. 9123!                                        Takii Naganuma Breeding Station, Naganuma-cho, Yubari-gun, Hokkaido,           1991-1992                                                                                   1991           1992                                                               Seed           Seed                                               Line      Area                                                                              produced    Area                                                                              produced                                        Line                                                                              combination                                                                              (m.sup.2)                                                                         (l)  Remarks                                                                               (m.sup.2)                                                                         (l)  Remarks                                    __________________________________________________________________________     AB 60To-AB × 2DR-B                                                                    -- --          9.0                                                                               2.0                                             B  2DR-B     -- --          4.5                                                                               0.7                                             F.sub.1                                                                           (60To-AB × 2DR-B) ×                                                          -- --          9.0                                                                               2.0  Field trial in                                62WeB-C                          the Netherlands                                                                (spring, 1993)                             __________________________________________________________________________

                                      TABLE 5                                      __________________________________________________________________________     Comparison of seed yields of F.sub.1  No. 9122! and F.sub.1  No. 9123!         Takii Naganuma Breeding Station, Naganuma-cho, Yubari-gun, Hokkaido            (Sowing: May 1, 1994)                                                                 Planting Susceptibility                                                                        Seed Seed Seed produc-                                  Spring, 1994                                                                          area                                                                               Number of                                                                           to blackleg                                                                           produced                                                                            produced                                                                            tion index                                    Cultivar                                                                              (m.sup.2)                                                                          plants                                                                              (++ - -)                                                                              (l)  (l/20 m.sup.2)                                                                      (%)                                           __________________________________________________________________________     No. 9122 (F.sub.1)                                                                    20.0                                                                               1800 ∓   9.4  9.4  98                                            No. 9123 (F.sub.1)                                                                    20.0                                                                               1800 ∓   9.6  9.6  100                                           Westar 10.0                                                                                320*                                                                               ++     1.2  2.4  25                                            OAC Triton                                                                            10.0                                                                                460*                                                                               +      2.2  4.4  47                                            __________________________________________________________________________      *: The high incidence of blackleg resulted in a decreased plant                population.                                                              

3. Breeding of a rape variety with introduced self-incompatibility

Starting with a line which was mainly spring type and double-low, breeding was performed for the purpose of introducing the self-incompatible genes of cabbages (B. oleracea, n=9) and Chinese cabbages (B. campestris, n=10).

The breeding processes of main 4 lines are described below.

1) Development of 59ReS^(a) ! (FIG. 5)

In order to introduce one of the self-incompatible genes of cabbage (factor a) into the spring type, double-low line 59Re! (B. napus, n=19), a synthetic napus (B. napus, n=19), viz. an amphidiploid, was developed from komatsuna (B. campestris, n=10) and cabbage (B. oleracea, n=9) and, further, after hybridization with nabana (B. campestris, n=10), crossing with a selected line of 59Re! was performed. The objective of crossing with nabana was as follows. Because of the use of a synthetic napus between green vernalization type cabbage and seed vernalization type komatsuna, it was considered necessary to bring them closer to spring types with weak low temperature response and strong day length response. Then, using a line selected with regard to spring habit and double-low characteristic, self-pollination was repeated 4 times to develop a rape line 59ReS^(a) ! having the self-incompatibility factor a from the synthetic napus. Moreover, reciprocal crossing of the line for reconversion to rape cytoplasm was also carried out.

2) Development of 59ReS^(b) ! (FIG. 6)

The first half of the breeding process was substantially the same as for the development of 59ReS^(a) ! and one of the self-incompatible genes of cabbages (factor b, different from factor a) was introduced. Here, using a selected line from rape line 59Re! as the pollen parent, crossing was carried out once and, then, using a selected line of 59Re! as the female parent, crossing was carried out twice. Thus, the conversion from komatsuna cytoplasm to rape cytoplasm was made to introduce the stability of rape phenotype and a line 59ReS^(b) ! of low glucosinolate content having factor b was obtained.

3) Development of 62WeS^(b) ! (FIG. 7)

By the same procedure as the development of 59ReS^(b) !, factor b was introduced into the spring type, double-low line 62We!.

4) Development of H-Bi-S^(d) ! (FIG. 8)

A synthetic napus was developed from komatsuna and cabbage as an amphidiploid and crossed with nabana, and its progeny was backcrossed with the spring type, double-low rape line 62We! twice, and then a line was developed by self-pollinating and selection. On the other hand, one line of synthetic napus obtained by cell fusion between a cabbage line ER159! and Chinese cabbage Green Rocket 70! was crossed with rape line LE112-82!, followed by crossing with a selected one from rape line 60To! to develop a crossing line.

These two lines were crossed to develop a double-low line having the factor d derived from cabbage ER159!, self-incompatibility and improved cold resistance.

4. Breeding of a rape line which has both a fertility restoring gene for male sterility and a self-incompatible gene

1) Development of H-Bi-S^(d) B! (FIG. 9)

By crossing three lines, viz. a line obtained by introducing a rape line IM!-derived fertility restoring gene for rape line MS-N1!-derived cytoplasm with male sterility into a selected one from rape line 59Re!, a selected crossing line between said synthetic napus and rape line 60To!, and a selected one from rape line EG1-83!, a double-low rape line H-Bi-S^(d) B! having a fertility restoring gene for male sterility and self-incompatibility factor d was developed.

5. Breeding of a line by using a combination of the male sterility with self-incompatibility in Brassicaceae plants other than rape

This breeding process is now described with reference to radish (Raphanus sativus, n=9) and karashina (mustard plant) (B. juncea, n=18).

1) Development of radish AB lines 62Z₅₅ -AB! and 62Z₅₆ -AB! (FIG. 10)

Referring to 62Z₅₅ -AB!, in case that a radish male sterile line R-5! was first crossed with the parent line Z₅₅ ! of an established radish F₁ variety which had homozygotically one incompatible gene (factor 5), in the next generation 1-2165! all the progeny was male-sterile. Therefore, using Z₅₅ ! as a maintainer, continuous backcrossing was carried out. In 1991, 3-70021! was subjected to CO₂ treatment for temporary overthrow of self-incompatibility and 62Z₅₅ -AB! was developed by mass seed production with bees for crossing.

As to 62Z₅₆ -AB!, the breeding process up to 3-70021! in 1991 was the same as for 62Z₅₅ -AB! but this 3-70021! was crossed with Z₆₆ ! which was substantially equivalent to Z₅₅ ! genetically but had a different self-incompatible gene. Because of the different self-incompatible gene, CO₂ treatment was unnecessary in this case. Another difference from 62Z₅₅ -AB! was that an increased seed yield was obtained because of the hybrid vigour due to crossing with Z₆₆ !.

2) Development of karashina (mustard plant) AB line 1PP-AB! (FIG. 11)

For the F₁ breeding of karashina (B. juncea, n=18) which is self-compatible, a karashina line 1PP-B! confirmed to act as a maintainer for the rape (B. napus, n=19) male sterile line 60To-AB! was selected and continuous backcrossing was initiated. For 60To-AB!, nucleus substitution using 1PP-B! was carried out.

3) Development of a radish self-compatible AB line OK-AB! (FIG. 12)

The radish self-compatible line OK! was found to be a maintainer providing all the progeny with male sterility for a male sterile line R-5! (genetically a cytoplasmic male sterile line) and development of a radish self-compatible male sterile line was started in 1987. As a result, OK-AB! was obtained in 1992.

The method of F₁ breeding by the combination of male sterility and self-incompatibility using the above lines is now described.

6. Production of F₁ seed using the female parent of a male sterile line introduced self-incompatibility and the male parent of a self-incompatible line (FIG. 13)

This experiment was performed on radish in which the production of F₁ seed is conventionally carried out mostly by four-way crossing utilizing self-incompatibility. Regarding radish, intra contamination is a frequent problem and, moreover, the number of seed grains per pod is small. Therefore, the seed production cost is high and a demand exists for cost reduction. The development of several lines in which the nucleus substitution of cytoplasmic male sterile line with the parent lines of F₁ was carried out, was already completed. As regards seed production of self-incompatible line, lines permitting seed production by carbon dioxide treatment were utilized.

1) Production of F₁ seed by single crossing (FIG. 14)

It was confirmed in 1987-1988 that the parent line Z_(55!) of F₁ which had already been developed acts as a maintainer (B line) for the male sterile line R-5! and continuous backcrossing with Z₅₅ ! was started. The 1991 3-70021! line with about 95% nucleus substitution (corresponding to msS₁ ! at top left in 1 of FIG. 13) was crossed with Z₅₅ ! (corresponding to S₁ !) by carbon dioxide treatment and as a result, 62Z₅₅ -AB! (4-2143) was obtained in 1992. This 62Z₅₅ -AB! corresponds to msS₁ ! in the center in 1 of FIG. 13. Using this line as the female parent, F₁ (corresponding to msS₁₃ ! in FIG. 13) was obtained by crossing it with TM₂₂ ! (corresponding to S₃ ! in 1 of FIG. 13) which was a separately developed parent line confirmed to have an excellent F₁ -combining ability with respect to Z₅₅ !.

2) Production of F₁ seed by three-way crossing (FIG. 15)

The process was substantially the same as the above production of F₁ seed by single crossing but was different in that, in 1991, the 3-70021! female parent was crossed with Z₆₆ ! which was substantially equivalent to Z₅₅ ! genetically but differed from the latter in the self-incompatible gene. In this case, because of the difference in incompatibility factor, CO₂ treatment was unnecessary. In 1992, 62Z₅₆ -AB! (4-2147) was obtained and F₁ was developed by crossing with TM₂₂ ! as in the single crossing described in 1). 62Z₅₆ -AB! corresponds to msS₁₂ ! in 2 of FIG. 13.

This procedure is different from the single crossing described above in 1) in that CO₂ treatment is not required and that the seed yield of 62Z₅₆ -AB! exceeds that of the single-crossed hybrid 62Z₅₅ -AB!.

3) Production of F₁ seed by four-way crossing (FIG. 16)

The female parent side was the same as that used in the three-way crossing in 2) but TM₁₁ !, a line which was genetically equivalent to TM₂₂ ! but had a different incompatibility factor, was added to the male parent side. TM₂₂ ! corresponds to S₃ ! in 3 of FIG. 13. Similarly, TM₁₁ ! corresponds to S₄ ! and TM₂₁ ! corresponds to S₃₄ !.

The foregoing is a description of the processes 1-3 of FIG. 13, taking radish as an example. In actual practice, for cabbages, Chinese cabbages, turnips, etc. which yield large amounts of seed per pod and are comparatively easy to increase seed yields, it is unnecessary to develop a line which is genetically equivalent but has a different incompatibility factor and the procedure 1) (single crossing) and procedure 2) (three-way crossing), both shown in FIG. 13, are suitable. For radish and other crops which are rather poor in seed yield, the procedure 3) (four-way crossing) is most suitable.

7. Production of F₁ seed by using the female parent of a male sterile line introduced self-incompatibility and the male parent of a self-incompatible or self-compatible line possessing fertility restoring gene (FIG. 17)

1) Production of F₁ seed by three-way crossing utilizing a male parent of a self-compatible line (FIGS. 18-21; FIG. 19 is sequential to the bottom of FIG. 18, FIG. 20 is sequential to the right of FIG. 18, and FIG. 21 is sequential to the bottom of FIG. 20 and the right of FIG. 19)

Referring to 1 of FIG. 17, msS₁ ! corresponds to 1993 5-32008!; S₂ ! corresponds to 1992 4-80151!; msS₁₂ ! corresponds to 1993 5-82407!; and rS_(f1) ! corresponds to 1992 4-80005!.

The male sterile line 60To-AB! (1991-3ND-42001) was crossed with 59ReS^(b) ! having a self-incompatible gene (factor b) 4 times since 1991 to obtain 5-32008! in 1993. This line was crossed with 59ReS^(a) ! (1992-4-80151) having a different self-incompatible gene (factor a) to develop the female parent of a male sterile line introduced self-incompatibility. Then, using the self-compatible male parent possessing fertility restoring gene WeB-C! (1992-4-80005), F₁ msS₁₂ ·rS_(f1) ! was obtained.

2) Production of F₁ seed by three-way crossing utilizing a male parent of a self-incompatible line (FIGS. 22-25. FIG. 23 is sequential to the bottom of FIG. 22; FIG. 24 is sequential to the right of FIG. 22; and FIG. 25 is sequential to the bottom of FIG. 24 and the right of FIG. 23).

Referring to 2 of FIG. 17, msS₁ !, S₂ ! and msS₁₂ ! correspond to the respective lines mentioned for the three-way crossing described in 1), and rS₃ ! in 2 of FIG. 17 corresponds to H-Bi-S^(d) B! (1992-4-84004). Because the male parent introduced self-incompatibility was used, the male parent produced no seed and omnibus cutting was possible at the production of F₁ seed, thus contributing to cost reduction.

3) Production of F₁ seed by four-way crossing (FIGS. 26-31. FIG. 27 is sequential to the bottom of FIG. 26; FIG. 28 is sequential to the right of FIG. 26; FIG. 29 is sequential to the bottom of FIG. 28 and the right of FIG. 27; FIG. 30 is sequential to the right of FIG. 28; and FIG. 31 is sequential to the bottom of FIG. 30 and the right of FIG. 29)

The female parent line was the same as used in the three-way crossings in 1) and 2) above, but a different male parent line was used. Referring to 3 of FIG. 17, rS₃ ! corresponds to H-Bi-S^(d) B! (1992-4-84004), rS₄ ! corresponds to H-En-S^(e) B! (1992-4-84010), and rS₃₄ ! corresponds to 1992 4-31201!. Thus, 1993 5-82407! was crossed with 1992 4-31201! to develop F₁. By the combination of some self-incompatibility factors, not only the seed production capacity of F₁ was increased but also the utilization of F₂ was facilitated. By this procedure, mass seed production and cost reduction can be realized.

8. Production of F₁ seed using the female parent of a male sterile line introduced self-compatibility and the male parent of a self-compatible line or the male parent of a self-incompatible line (FIG. 32)

Karashina (B. juncea, n=18) and radish (R. sativus, n=9) were used. While karashina is self-compatible, radish may be self-compatible or self-incompatible.

1) Production of F₁ seed by utilizing the male parent of a self-compatible line (FIG. 33)

As it was found that the 1PP-B! line of karashina (n=18) acts as a maintainer for 60To-AB!, i.e. a male sterile line of rape (n=19), nucleus substitution is performed by continuous backcrossing. Backcrossing through 2-3 generations from the 1993 5-2256! is necessary. In this way the karashina AB line 1PP-AB! is obtained. Then, using 62CbAe-C! under developing, for instance, as C line, F₁ breeding is performed.

Referring to 1 of FIG. 32, msS_(f1) ! corresponds to 1PP-AB! (progeny of 1993-5-2256) and S_(f2) ! corresponds to 62CbAe-C! (progeny of 1993-5-102).

2) Production of F₁ seed by utilizing the male parent of a self-incompatible line: karashina (FIG. 34)

Referring to 2 of FIG. 32, msS_(f1) ! corresponds to 1PP-AB! (progeny of 1993-5-2256) and S₁ ! corresponds to 62CBS^(a) ! (progeny of 1993-5-704). This 62CBS^(a) ! is a line obtained by crossing 62Cb! with a Chinese cabbage line (B. campestris, n=10) to introduce a self-incompatible gene and given n=18 chromosome number and improved characters through selection and breeding. F₁ is developed using this line as the male parent of a self-incompatible line.

3) Production of F₁ seed by utilizing the male parent of a self-incompatible line: radish (FIG. 35)

When a self-compatible radish line OK! (1987-62-4582) was crossed with R-5! (1987-62-2072) having cytoplasm with male sterility, it was found that OK! acts as a maintainer. Therefore, OK-AB! (1992-4-2155) was developed by continuous backcrossing. This line was crossed with the established parent line TM₁₂ ! to develop F₁ (corresponding to msS_(f1) S₁ ! in 2 of FIG. 32).

9. Production of F₁ seed by utilizing the female parent of a male sterile line introduced self-compatibility and the male parent of a self-incompatible line possessing fertility restoring gene (FIG. 36)

1) Production of F₁ seed by single crossing (FIGS. 37-40. FIG. 38 is sequential to the bottom of FIG. 37; FIG. 39 is sequential to the right of FIG. 37; and FIG. 40 is sequential to the bottom of FIG. 39 and the right of FIG. 38).

The line corresponding to msS_(f1) ! in 1 of FIG. 36 is 60To-AB! (1992-4-92005) and the line corresponding to rS₁ ! is H-Bi-S^(d) B! (1992-4-84004). Because of the introduction of self-incompatibility into the male parent line, omnibus reaping of female and male parents in the production of F₁ seed was feasible.

2) Production of F₁ seed by three-way crossing (FIGS. 41-44. FIG. 42 is sequential to the bottom of FIG. 41; FIG. 43 is sequential to the right of FIG. 41; and FIG. 44 is sequential to the bottom of FIG. 43 and the right of FIG. 42)

Referring to 2 of FIG. 36, msS_(f1) ! corresponds to 60To-AB! (1991-3ND-42001); S_(f2) ! corresponds to 2DR-B! (1991-3N-40514); msS_(f12) ! corresponds to 1992 4-82072!; and rS₁ ! corresponds to H-Bi-S^(d) B! (1992-4-84004). For enhancement of disease resistance, lodging resistance and seed yield, 2DR-B! possessing such characteristics was introduced.

3) Production of F₁ seed by four-way crossing (FIGS. 45-50. FIG. 46 is sequential to the bottom of FIG. 45; FIG. 47 is sequential to the right of FIG. 45; FIG. 48 is sequential to the bottom of FIG. 47 and the right of FIG. 46; FIG. 49 is sequential to the right of FIG. 47; and FIG. 50 is sequential to the bottom of FIG. 49 and the right of FIG. 48)

The above process is different from the three-way crossing described in 2) in that H-En-S^(e) B! (1992-40) corresponding to rS₂ ! in 3 of FIG. 36 was introduced into the male parent side. By this procedure, the mass production of rS₁₂ ! was facilitated and the utility of F₂ was enhanced. 

We claim:
 1. A method of inhibiting selfing in breeding crop plants in the family Brassicaceae to produce F₁ hybrid seed which comprises the steps of crossing a male-sterile self-incompatible line as a female parent with a self-incompatible plant as the male parent to produce seed in said female parent, and harvesting the seed produced.
 2. A method of inhibiting selfing in breeding crop plants in the family Brassicaceae to produce F₁ hybrid seed which comprises the steps of crossing a male-sterile self-incompatible line as a female parent with a male parent selected from the group consisting of a self-incompatible plant possessing fertility restoring gene as the male parent and a self-compatible plant possessing fertility restoring gene as the male parent to produce seed in said female parent, and harvesting the seed produced. 